Synthesis of PNA FIT Probes

The PNA FIT probes contain a single cyanine dye that belongs to the thiazole orange (TO) family of intercalator dyes. The dye replaces a canonical nucleobase and, thereby, serves as a fluorescent base surrogate. PNA FIT probes respond to changes of the viscosity around the environmentally sensitive TO dye. 

The simultaneous imaging of two viral mRNA targets requires two differently colored probes. This calls for two spectrally resolvable dyes. Among the various members of the TO family of dyes, we selected the quinolinium-based TO (maximal fluorescence emission fg, (max) = 530 nm) and the pyridinium-based pyridinium benzothiazole (BO) dye (fg, (max)=487nm). Carboxymethylated versions of both dyes were introduced as base surrogates in FIT probes 

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